Nicking and Extension Amplification Reaction for the Exponential Amplification of Nucleic Acids

ABSTRACT

The invention is in general directed to the rapid exponential amplification of short DNA or RNA sequences at a constant temperature.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/778,018, filed Jul. 14, 2007, the entire contents of which are herebyincorporated.

FIELD OF THE INVENTION

The invention is in general directed to the rapid exponentialamplification of short DNA or RNA sequences at a constant temperature.

BACKGROUND

The field of in vitro diagnostics is quickly expanding as the need forsystems that can rapidly detect the presence of harmful species ordetermine the genetic sequence of a region of interest is increasingexponentially. Current molecular diagnostics focus on the detection ofbiomarkers and include small molecule detection, immuno-based assays,and nucleic acid tests. The built-in specificity between twocomplementary nucleic acid strands allows for fast and specificrecognition using unique DNA or RNA sequences, the simplicity of whichmakes a nucleic acid test an attractive prospect. Identification ofbacterial and viral threat agents, genetically modified food products,and single nucleotide polymorphisms for disease management are only afew areas where the advancement of these molecular diagnostic toolsbecomes extremely advantageous. To meet these growing needs, nucleicacid amplification technologies have been developed and tailored tothese needs of specificity and sensitivity.

Historically, the most common amplification technique is the polymerasechain reaction (PCR), which has in many cases become the gold standardfor detection methods because of its reliability and specificity. Thistechnique requires the cycling of temperatures to proceed through thesteps of denaturation of the dsDNA, annealing of short oligonucleotideprimers, and extension of the primer along the template by athermostable polymerase. Though many new advances in engineering havesuccessfully shortened these reaction times to 20-30 minutes, there isstill a steep power requirement to meet the needs of these thermocyclingunits.

Various isothermal amplification techniques have been developed tocircumvent the need for temperature cycling. From this demand, both DNAand RNA isothermal amplification technologies have emerged.

Transcription-Mediated Amplification (TMA) employs a reversetranscriptase with RNase activity, an RNA polymerase, and primers with apromoter sequence at the 5′ end. The reverse transcriptase synthesizescDNA from the primer, degrades the RNA target, and synthesizes thesecond strand after the reverse primer binds. RNA polymerase then bindsto the promoter region of the dsDNA and transcribes new RNA transcriptswhich can serve as templates for further reverse transcription. Thereaction can produce a billion fold amplification in 20-30 minutes. Thissystem is not as robust as other DNA amplification techniques and istherefore, not a field-deployable test due to the ubiquitous presence ofRNAases outside of a sterile laboratory. This amplification technique isvery similar to Self-Sustained Sequence Replication (3SR) and NucleicAcid Sequence Based Amplification (NASBA), but varies in the enzymesemployed.

Single Primer Isothermal Amplification (SPIA) also involves multiplepolymerases and RNaseH. First, a reverse transcriptase extends achimeric primer along an RNA target. RNaseH degrades the RNA target andallows a DNA polymerase to synthesize the second strand of cDNA. RNaseHthen degrades a portion of the chimeric primer to release a portion ofthe cDNA and open a binding site for the next chimeric primer to bindand the amplification process proceeds through the cycle again. Thelinear amplification system can amplify very low levels of RNA target inroughly 3.5 hrs.

The Q-Beta replicase system is a probe amplification method. A proberegion complementary to the target of choice is inserted into MDV-1 RNA,a naturally occurring template for Q-Beta replicase. Q-Beta replicatesthe MDV-1 plasmid so that the synthesized product is itself a templatefor Q-Beta replicase, resulting in exponential amplification as long asthe there is excess replicase to template. Because the Q-Betareplication process is so sensitive and can amplify whether the targetis present or not, multiple wash steps are required to purge the sampleof non-specifically bound replication plasmids. The exponentialamplification takes approximately 30 minutes; however, the total timeincluding all wash steps is approximately 4 hours.

Numerous isothermal DNA amplification technologies have been developedas well. Rolling circle amplification (RCA) was developed based on thenatural replication of plasmids and viruses. A primer extends along acircular template resulting in the synthesis of a single-stranded tandemrepeat. Capture, washing, and ligation steps are necessary topreferentially circularize the template in the presence of target andreduce background amplification. Ramification amplification (RAM) addscascading primers for additional geometric amplification. This techniqueinvolves amplification of non-specifically sized strands that are eitherdouble or single-stranded.

Helicase-dependent amplification (HDA) takes advantage of a thermostablehelicase (Tte-UvrD) to unwind dsDNA to create single-strands that arethen available for hybridization and extension of primers by polymerase.The thermostable HDA method does not require the accessory proteins thatthe non-thermostable HDA requires. The reaction can be performed at asingle temperature, though an initial heat denaturation to bind theprimers generates more product. Reaction times are reported to be over 1hour to amplify products 70-120 base pairs in length.

Loop mediated amplification (LAMP) is a sensitive and specificisothermal amplification method that employs a thermostable polymerasewith strand displacement capabilities and four or more primers. Theprimers are designed to anneal consecutively along the target in theforward and reverse direction. Extension of the outer primers displacesthe extended inner primers to release single strands. Each primer isdesigned to have hairpin ends that, once displaced, snap into a hairpinto facilitate self-priming and further polymerase extension. Additionalloop primers can decrease the amplification time, but complicates thereaction mixture. Overall, LAMP is a difficult amplification method tomultiplex, that is, to amplify more than one target sequence at a time,although it is reported to be extremely specific due to the multipleprimers that must anneal to the target to further the amplificationprocess. Though the reaction proceeds under isothermal conditions, aninitial heat denaturation step is required for double-stranded targets.Amplification proceeds in 25 to 50 minutes and yields a ladder patternof various length products.

Strand displacement amplification (SDA) was developed by Walker et. al.in 1992. This amplification method uses two sets of primers, a stranddisplacing polymerase, and a restriction endonuclease. The bumperprimers serve to displace the initially extended primers to create asingle-strand for the next primer to bind. A restriction site is presentin the 5′ region of the primer. Thiol-modified nucleotides areincorporated into the synthesized products to inhibit cleavage of thesynthesized strand. This modification creates a nick site on the primerside of the strand, which the polymerase can extend. This approachrequires an initial heat denaturation step for double-stranded targets.The reaction is then run at a temperature below the melting temperatureof the double-stranded target region. Products 60 to 100 bases in lengthare usually amplified in 30-45 minutes using this method.

These and other amplification methods are discussed in, for example,VanNess, J, et al., PNAS 2003. Vol. 100, no 8, p 4504-4509; Tan, E., etal., Anal. Chem. 2005, 77, 7984-7992; Lizard, P., et al., NatureBiotech. 1998, 6, 1197-1202; Notomi, T., et al., NAR 2000, 28, 12, e63;and Kurn, N., et al., Clin. Chem. 2005, 51:10, 1973-1981. Otherreferences for these general amplification techniques include, forexample, U.S. Pat. Nos. 7,112,423; 5,455,166; 5,712,124; 5,744,311;5,916,779; 5,556,751; 5,733,733; 5,834,202; 5,354,668; 5,591,609;5,614,389; 5,942,391; and U.S. patent publication numbers US20030082590;US20030138800; US20040058378; and US20060154286.

There is a need for a quicker method of amplification of single-strandedand double-stranded nucleic acid target sequences that can be performedwithout temperature cycling and that is suitable for shorter targetsequences.

SUMMARY

Provided herein are methods of amplifying nucleic acid target sequencesthat rely on nicking and extension reactions and amplify shortersequences in a quicker timeframe than traditional amplificationreactions, such as, for example, strand displacement amplificationreactions. Embodiments of the invention include, for example, reactionsthat use only two templates to prime, one or two nicking enzymes, and apolymerase, under isothermal conditions. In exemplary embodiments, thepolymerase and the nicking enzyme are thermophilic, and the reactiontemperature is significantly above the melting temperature of thehybridized target region. The nicking enzyme nicks only one strand in adouble-stranded duplex, so that incorporation of modified nucleotides isnot necessary as it is in strand displacement. An initial heatdenaturation step is not required for the methods of the presentinvention. Due to the simplicity of the reaction, in exemplaryembodiments, the reaction is very easy to perform and can amplify20-30mer products 10⁸ to 10¹⁰ fold from genomic DNA in 2.5 to 10minutes. Furthermore, in other exemplary embodiments, the method is ableto amplify RNA without a separate reverse transcription step.

Thus, provided in a first embodiment of the present invention is amethod for amplifying a double-stranded nucleic acid target sequence,comprising contacting a target DNA molecule comprising a double-strandedtarget sequence having a sense strand and an antisense strand, with aforward template and a reverse template, wherein said forward templatecomprises a nucleic acid sequence comprising a recognition region at the3′ end that is complementary to the 3′ end of the target sequenceantisense strand; a nicking enzyme site upstream of said recognitionregion, and a stabilizing region upstream of said nicking enzyme site;said reverse template comprises a nucleotide sequence comprising arecognition region at the 3′ end that is complementary to the 3′ end ofthe target sequence sense strand, a nicking enzyme site upstream of saidrecognition region, and a stabilizing region upstream of said nickingenzyme site; providing a first nicking enzyme that is capable of nickingat the nicking enzyme site of said forward template, and does not nickwithin said target sequence; providing a second nicking enzyme that iscapable of nicking at the nicking enzyme site of said reverse templateand does not nick within said target sequence; and providing a DNApolymerase; under conditions wherein amplification is performed bymultiple cycles of said polymerase extending said forward and reversetemplates along said target sequence producing a double-stranded nickingenzyme site, and said nicking enzymes nicking at said nicking enzymesites, producing an amplification product.

In certain embodiments of the invention, the DNA polymerase is athermophilic polymerase. In other examples of the invention, thepolymerase and said nicking enzymes are stable at temperatures up to 37°C., 42° C., 60° C., 65° C., 70° C., 75° C., 80° C., or 85° C. In certainembodiments, the polymerase is stable up to 60° C. The polymerase may,for example, be selected from the group consisting of Bst (largefragment), 9° N, VentR® (exo-) DNA Polymerase, Therminator, andTherminator II.

The nicking enzyme may, for example, nick upstream of the nicking enzymebinding site, or, in exemplary embodiments, the nicking enzyme may nickdownstream of the nicking enzyme binding site. In certain embodiments,the forward and reverse templates comprise nicking enzyme sitesrecognized by the same nicking enzyme and said first and said secondnicking enzyme are the same. The nicking enzyme may, for example, beselected from the group consisting of Nt.BspQI, Nb.BbvCi, Nb.BsmI,Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.BpulOI,and Nt.Bpul0I.

In certain aspects of the present invention, the target sequencecomprises from 1 to 5 nucleotides more than the sum of the nucleotidesof said forward template recognition region and said reverse templaterecognition region.

The DNA molecule may be, for example, genomic DNA. The DNA molecule maybe, for example, selected from the group consisting of plasmid,mitochondrial, and viral DNA. In certain embodiments, the forwardtemplate is provided at the same concentration as the reverse template.In other examples, the forward template is provided at a ratio to thereverse template at the range of ratios of 1:100 to 100:1.

In other examples of the invention, the method further comprises the useof a second polymerase. The amplification may be, for example, conductedat a constant temperature. This temperature may be, for example, between54° C. and 60° C. As to the length of time for the reaction to takeplace, in certain examples, the amplification reaction is held atconstant temperature for 1 to 10 minutes.

The present invention further comprises detecting the amplificationproduct, for example, by a method selected from the group consisting ofgel electrophoresis, mass spectrometry, SYBR I fluorescence, SYBR IIfluorescence, SYBR Gold, Pico Green, TOTO-3, intercalating dyedetection, FRET, molecular beacon detection, surface capture, capillaryelectrophoresis, incorporation of labeled nucleotides to allow detectionby capture, fluorescence polarization, and lateral flow capture. Theamplification products may be, for example, detected using a solidsurface method, for example, where at least one capture probe isimmobilized on the solid surface that binds to the amplified sequence.

The present invention may be used for multiplex amplification. Thus, forexample, in certain embodiments of the present invention at least twotarget sequences are capable of being amplified. By “capable of beingamplified” is meant the amplification reaction comprises the appropriatetemplates and enzymes to amplify at least two target sequences. Thus,for example, the amplification reaction may be prepared to detect atleast two target sequences, but only one of the target sequences mayactually be present in the sample being tested, such that both sequencesare capable of being amplified, but only one sequence is. Or, where twotarget sequences are present, the amplification reaction may result inthe amplification of both of the target sequences. The multiplexamplification reaction may result in the amplification of one, some, orall, of the target sequences for which it comprises the appropriatetemplates and enzymes.

At least one of the templates, for example, may comprise a spacer, ablocking group, or a modified nucleotide.

Also provided as an embodiment of the present invention is a method foramplifying a single-stranded nucleic acid target sequence, comprisingcontacting a target nucleic acid comprising a single-stranded targetsequence with a reverse template, wherein said reverse templatecomprises a nucleotide sequence comprising a recognition region at the3′ end that is complementary to the 3′ end of the target sequence, anicking enzyme site upstream of said recognition region, and astabilizing region upstream of said nicking enzyme site; providing afirst nicking enzyme that is capable of nicking at the nicking enzymesite of said reverse template, and does not nick within said targetsequence; providing a DNA polymerase under conditions wherein saidpolymerase extends said reverse template along said target sequence;contacting said extended reverse template with a forward template,wherein said forward template comprises comprising a recognition regionat the 3′ end that is identical to the 5′ end of the target sequence anicking enzyme site upstream of said recognition region, and astabilizing region upstream of said nicking enzyme site; providing asecond nicking enzyme that is capable of nicking at the nicking enzymesite of said forward template and does not nick within said targetsequence; under conditions wherein amplification is performed bymultiple cycles of said polymerase extending said forward and reversetemplates along said target sequence producing a double-stranded nickingenzyme site, and said nicking enzymes nicking at said nicking enzymesites, producing an amplification product.

Those of ordinary skill in the art understand that the examplespresented herein relating to the amplification of a double-strandednucleic acid target sequence and the detection of the amplified productalso apply to the amplification of a single-stranded nucleic acid targetsequence and the detection of the amplified product. Further, inexamples of the present invention, the target sequence may be, forexample, RNA, for example, but not limited to, messenger RNA, viral RNA,microRNA, a microRNA precursor, or siRNA. In exemplary embodiments ofthe present invention, the polymerase comprises reverse transcriptionactivity. In yet other examples of the present invention, the targetsequence is DNA, such as, for example, genomic DNA, or for example, thetarget sequence is selected from the group consisting of plasmid,mitochondrial, and viral nucleic acid.

Where the method may comprise the use of more than one polymerase, inexemplary embodiments at least one of the polymerases comprises reversetranscriptase activity.

In other embodiments of the present invention, a set of oligonucleotidetemplates is provided, comprising a first template for nucleic acidamplification, comprising a recognition region at the 3′ end that iscomplementary to the 3′ end of a target sequence antisense strand; anicking enzyme site upstream of said recognition region; and astabilizing region upstream of said nicking enzyme site; and a secondtemplate for nucleic acid amplification, comprising a recognition regionat the 3′ end that is identical to the 5′ of said target sequenceantisense strand; a nicking enzyme site upstream of said recognitionregion; and a stabilizing region upstream of said nicking enzyme site;wherein said target sequence comprises from 1 to 5 spacer bases betweensaid 3′ end of the antisense strand and said 5′ end of said antisensestrand that do not bind to either template.

In yet other embodiments, a kit is provided for following the methods ofthe present invention for nucleic acid amplification, comprising a DNApolymerase; a first template for nucleic acid amplification, comprisinga recognition region at the 3′ end that is complementary to the 3′ endof a target sequence antisense strand; a nicking enzyme site upstream ofsaid recognition region; and a stabilizing region upstream of saidnicking enzyme site; a second template for nucleic acid amplification,comprising a recognition region at the 3′ end that is complementary tothe 3′ end of a target sequence sense strand; a nicking enzyme siteupstream of said recognition region; and a stabilizing region upstreamof said nicking enzyme site; one or two thermostable nicking enzymes,wherein either one enzyme is capable of nicking at the nicking enzymesite of said first and said second templates, or a first enzyme iscapable of nicking at the nicking enzyme site of said first primer and asecond enzyme is capable of nicking at the enzyme site of said secondprimer.

The kit may, for example, provide said polymerase, nicking enzymes, andtemplates in a container. The kit may provide, for example, saidpolymerase, nicking enzymes, and templates in two containers. In certainexamples, the polymerase and nicking enzymes are in a first container,and said templates are in a second container. In certain examples, thepolymerase and nicking enzymes are lyophilized. The kit may, forexample, further comprise instructions for following the amplificationmethods of the present invention. The kit may, for example, furthercomprise a cuvette. The kit may, for example, further comprise a lateralflow device or dipstick. The lateral flow device or dipstick may, forexample, further comprise a capture probe, wherein said capture probebinds to amplified product. The kit may, for example, further comprise adetector component selected from the group consisting of a fluorescentdye, colloidal gold particles, latex particles, a molecular beacon, andpolystyrene beads. In other examples, at least one of the templates ofthe kit comprises a spacer, blocking group, or a modified nucleotide.

Deoxynucleoside triphosphates (dNTPs) are included in the amplificationreaction. One or more of the dNTPs may be modified, or labeled, asdiscussed herein. Nucleotides are designated as follows. Aribonucleoside triphosphate is referred to as NTP or rNTP; N can be A,G, C, U or m5U to denote specific ribonucleotides. Deoxynucleosidetriphosphate substrates are indicated as dNTPs, where N can be A, G, C,T, or U. Throughout the text, monomeric nucleotide subunits may bedenoted as A, G, C, or T with no particular reference to DNA or RNA.

In another embodiment, a method is provided for nucleic acidamplification comprising forming a mixture of a target nucleic acidcomprising a double-stranded target sequence having a sense strand andan antisense strand; a forward template comprising a nucleic acidsequence comprising a recognition region at the 3′ end that iscomplementary to the 3′ end of the target sequence antisense strand; anicking enzyme site upstream of said recognition region, and astabilizing region upstream of said nicking enzyme site; a reversetemplate comprising a nucleotide sequence comprising a recognitionregion at the 3′ end that is complementary to the 3′ end of the targetsequence sense strand, a nicking enzyme site upstream of saidrecognition region and a stabilizing region upstream of said nickingenzyme site; a first nicking enzyme that is capable of nicking at thenicking enzyme site of said forward template, and does not nick withinsaid target sequence; a second nicking enzyme that is capable of nickingat the nicking enzyme site of said reverse template and does not nickwithin said target sequence; and a thermophilic polymerase underconditions wherein amplification is performed by multiple cycles of saidpolymerase extending said forward and reverse templates along saidtarget sequence producing a double-stranded nicking enzyme site, andsaid nicking enzymes nicking at said nicking enzyme sites, producing anamplification product. In certain embodiments, the nicking enzyme siteson the forward and reverse templates are recognized by the same nickingenzyme, and only one nicking enzyme is used for the reaction.

In another embodiment, a method is provided for nucleic acidamplification comprising forming a mixture of a target nucleic acidcomprising a single-stranded target sequence; a reverse template,wherein said reverse template comprises a nucleotide sequence comprisinga recognition region at the 3′ end that is complementary to the 3′ endof the target sequence, a nicking enzyme site upstream of saidrecognition region, and a stabilizing region upstream of said nickingenzyme site; a first nicking enzyme that is capable of nicking at thenicking enzyme site of said reverse template, and does not nick withinsaid target sequence; a thermophilic polymerase under conditions whereinsaid polymerase extends said reverse template along said targetsequence; a forward template, wherein said forward template comprises anucleic acid sequence comprising a recognition region at the 3′ end thatis identical to the 5′ end of the target sequence; and a second nickingenzyme that is capable of nicking at the nicking enzyme site of saidforward template and does not nick within said target sequence; underconditions wherein amplification is performed by multiple cycles of saidpolymerase extending said forward and reverse templates along saidtarget sequence producing a double-stranded nicking enzyme site, andsaid nicking enzymes nicking at said nicking enzyme sites, producing anamplification product. In certain embodiments, the nicking enzyme siteson the forward and reverse templates are recognized by the same nickingenzyme, and only one nicking enzyme is used for the reaction.

In other embodiments of the invention are provided methods for theseparation of amplified nucleic acids obtained by the amplificationmethods of the invention. In yet further embodiments of the inventionare provided methods for detecting and/or analyzing the amplifiednucleic acids obtained by the amplification methods of the invention,including, for example, methods using SYBR I, II, SYBR Gold, Pico Green,TOTO-3, and most intercalating dyes, molecular beacons, FRET, surfacecapture using immobilized probes with fluorescence, electrochemical, orcolorimetric detection, mass spectrometry, capillary electrophoresis,the incorporation of labeled nucleotides to allow detection by captureor fluorescence polarization, lateral flow, and other methods involvingcapture probes. Methods using capture probes for detection include, forexample, the use of a nucleic acid molecule (the capture probe)comprising a sequence that is complementary to the amplified productsuch that the capture probe binds to amplified nucleic acid. Thereaction may, for example, further comprise an antibody directed againsta molecule incorporated into or attached to the capture probe. Or, forexample, the capture probe, or a molecule that binds to the captureprobe, may incorporate, for example, an enzyme label, for example,peroxidase, alkaline phosphatase, or beta-galactosidase, a fluorescentlabel, such as, for example, fluorescein or rhodamine, or, for example,other molecules having chemiluminescent or bioluminescent activity. Theembodiments of the present invention also comprise combinations of thesedetection and analysis methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D are graphic drawings depicting mechanisms of the reactions ofthe present invention. FIG. 1D is a legend for FIG. 1.

FIG. 2. 20% polyacrylamide gel of reaction products from a DNA NEARassay.

The NEAR reaction was run for 2.5 minutes at 56° C., then heat denaturedat 94° C. for 4 minutes. Six μL of the reaction was run on a 20%polyacrylamide gel at 160V for ˜2.5 hrs. The gel was stained with SYBRII gel stain. Lane 1: NEAR reaction no target control for 25mer assay.Lane 2: NEAR reaction no target control for 27mer assay. Lane 3: NEARreaction for 25mer assay with 3.5E+5 copies of genomic Bacillus subtilisDNA. Lane 4: NEAR reaction for 27mer assay with 1.1E+6 copies of genomicBacillus subtilis DNA.

FIG. 3. 20% polyacrylamide gel of reaction products from an RNA NEARassay.

The NEAR reaction was run for 12 minutes at 56° C., then heat denaturedat 94° C. for 4 minutes. Six μL of the reaction was run on a 20%polyacrylamide gel at 160V for −2.5 hrs. The gel was stained with SYBRII gel stain. Lane 1 & 2: NEAR reaction for 25mer assay with 1E+6 copiesof Ebola Armored RNA (Ambion). Lane 3 & 4: NEAR reaction no targetcontrol for 25mer assay. 25mer reaction products are outlined in thewhite box.

FIGS. 4A-B. Mass Spectrum of Bacillus anthracis DNA assay products.

A) 0 copies of target or B) 5E+5 copies of genomic DNA added to the NEARreaction. The NEAR reaction was run for 10 minutes, then heat denaturedat 94° C. for 4 minutes. Ten micro liters of sample was injected intothe LC/ESI-MS. The (−4) charge state of the 26mer product and itscomplementary sequence are outlined in a black box. The smaller adjacentpeaks are the sodium adducts of the main product.

FIGS. 5A-C. Mass Spectrum of MS2 genomic RNA assay products.

A) 0 copies of target, B) 1E+6 copies of MS2 genomic RNA, or C) 1E+6copies of synthetic target DNA added to the NEAR reaction. The NEARreaction was run for 10 minutes, then heat denatured at 94° C. for 4minutes. Ten micro liters of sample was injected into the LC/ESI-MS. The(−4) charge state of the 27mer product and its complement sequence areoutlined in a black box. The smaller adjacent peaks are the sodiumadducts of the main product.

FIG. 6. Real-time detection of NEAR assay amplification usingintercalating fluorescent dyes.

Real-time amplification of Yersinia pestis genomic DNA at 500 copies(squares) compared to the no target control (NTC, open triangles). Thereaction was run for 10 minutes at 58° C. and monitored by the real-timefluorescence with SYBR II (n=5).

FIG. 7. Real-time detection of NEAR assay amplification usingfluorescence resonance energy transfer (FRET).

Real-time amplification of Yersinia pestis synthetic DNA at 10,000copies (squares) compared to the no target control (NTC, opentriangles). The reaction was run for 10 minutes at 57° C., n=3.

FIG. 8. Francisella tularensis assay amplification detected in real-timeusing molecular beacons.

Either 0 copies (open triangles) or 1E+5 copies (squares) were added tothe reaction mix and run for 10 minutes at 57.5° C.

FIG. 9. False alarm rate testing results comparing average AVC values.

Error bars denote one standard deviation. Bacillus subtilis NEAR assayswere run for 10 min at 55° C. in the presence and absence of Bacillussubtilis genomic DNA. Enzymes were heat denatured at 94° C. for 4 min. A10 μL sample was injected into the LC/ESI-MS and the area under thecurve (AUC) of the product peaks were analyzed. True Positives contained10,000 copies of Bacillus subtilis along with 990,000 copies of nearneighbor (Bacillus thuringiensis). True Negatives contained 10,000copies of E. coli with 990,000 copies of near neighbor, and waternegatives contained no DNA as a control.

FIG. 10. Replication study of the NEAR Assay using molecular beacondetection with different operators performing the experiments on twodifferent days.

The NEAR reaction was run for 10 minutes at 57.5° C. (in the presenceand absence of 500 copies of Francisella tularensis genomic DNA) with a4 min heat kill at 94° C. 300 nM molecular beacon was added andmonitored at 45, 50, and 57° C. (n=24).

FIG. 11. Sensitivity of the NEAR reaction using molecular beacondetection.

The NEAR assay was run for 10 minutes 57.5° C. The reaction was stoppedwith a 4 min heat denaturation step at 94° C. 300 nM molecular beaconwas added and the fluorescence was monitored at 57.5° C. (n=3).Fluorescence was monitored for beacon opening in the presence NEARreactions amplified with 1E+6, 5E+5, 5E+4, 5E+2, 50, and 0 (NTC) inputcopies of Francisella tularensis genomic DNA, and compared to thebackground fluorescence of the beacon alone (MB).

FIG. 12. Final concentration of amplified products in the NEAR reaction.

The NEAR reaction was run for 10 min at 55° C. with varying copies ofBacillus subtilis genomic DNA. The reaction was stopped with a heatdenaturation step at 94° C. for 4 minutes. A 10 μL sample was injectedinto the LC/ESI-MS and the AUC of the product peak at 1944 Daltons wasanalyzed and compared to a standard curve.

FIG. 13. Correlation of the input RNA target copy number to the finalconcentration of amplified products.

The Ebola NEAR assay was run for 12 min at 55° C. with varying copies ofsynthetic RNA corresponding to the Ebola genome DNA. The reaction wasstopped with a heat denaturation step at 94° C. for 4 minutes. A 10 μLsample was injected into the LC/ESI-MS and the AUC of the product peakat 1936 Daltons was analyzed and compared to the standard curve of AUCvalues. (n=3)

FIG. 14. Mass spec product analysis demonstrating NEAR reactionspecificity.

The Bacillus anthracis NEAR reaction was run in the presence of adilution of copies of Bacillus thuringiensis for 10 min at 56° C. (n=3),then heat denatured at 94° C. for 4 minutes. A 10 μL sample was injectedinto the LC/ESI-MS and AUC values of product peaks analyzed.

FIG. 15. The effect of an interferent panel on the NEAR amplification.

Bacillus subtilis NEAR DNA reactions were run for 10 min at 55° C. andheated to 94° C. for 4 minutes to stop the reaction. Reactions were runin triplicate in the presence 1E+5 copies of Bacillus subtilis genomicDNA (“_(—)1E+5”) or with no target DNA present (“_(—)0”). Sample x isthe control assay with no interferent added. Interferents A through Fwere added at 50% reaction volume to the Bacillus subtilis assay. TheAUC of mass spec product peaks were analyzed using a two-way ANOV A andBonferroni t-test. (Key: A=none; B=House dust, skim milk; e=AZ testdust, humic acid; D=Diesel soot; E=Skim milk; F=Mold spores)

FIG. 16. Gel electrophoresis results for the Bacillus subtilis/Bacillusanthracis DNA duplex reaction.

The NEAR reaction including templates for both Bacillus subtilis (Bs)and Bacillus anthracis (Ba) assays was run in the absence of target DNA(negative), in the presence of Bacillus subtilis only (positive for27mer product), and in the presence of both Bacillus subtilis andBacillus anthracis (positive for 27mer and 25mer product respectively).The target copy number used in this assay was 500,000 copies. The assaywas run for 10 min at 57° C. Templates varied in concentration betweenthe assays to control the amplification (100 nM for Bacillus anthracisand 50 nM for Bacillus subtilis). Samples were run on a 20%polyacrylamide gel at 160 V for ˜2 hours. The gel was stained with SYBRII fluorescent dye and imaged. The fluorescent bands were quantitatedand analyzed as the integrated optical density (IOD) (n=8).

FIG. 17. Specificity results for the Bacillus subtilis/Bacillusanthracis DNA duplex reaction shown by gel electrophoresis.

The NEAR reaction including templates for both a Bacillus subtilis (Bs)and Bacillus anthracis (Ba) assay was run in the absence of target DNA(negative), in the presence of Bacillus subtilis only (27mer product),and in the presence of both Bacillus subtilis and Bacillus anthracis(27mer and 25mer product respectively). The target copy number for eachgenome present in this assay was 500,000 copies. All reactions contained500,000 copies of Bacillus thuringiensis as clutter. Templates varied inconcentration between the assays to control the amplification. The assaywas run for 10 min at 57° C., heat denatured at 94° C. for 4 min, and 6μL was loaded on to a 20% gel run at 160 V for ˜2 hours. The gel wasstained with SYBR II fluorescent dye and imaged. The fluorescent bandswere quantitated and analyzed as the integrated optical density (IOD).

FIG. 18. Gel electrophoresis results for the MS2/Eboia RNA duplexreaction.

The NEAR reaction including templates for both a MS2 and Ebola assay wasrun in the absence of target RNA (negative, lanes 2-5), in the presenceof MS2 only (27mer product, lanes 6 and 7), and in the presence of bothMS2 and Ebola RNA (27mer and 25mer product respectively, lanes 8 and 9).The target copy number used in this assay was 1E+6 copies. The assay wasrun for 10 min at 57° C. Templates varied in concentration between theassays to control the amplification. Samples were run on a 20%polyacrylamide gel at 160 V for ˜2.5 hours. The gel was stained withSYBR II fluorescent dye and imaged. The fluorescent bands werequantitated and analyzed as the integrated optical density (IOD).

FIG. 19. Mass spec analysis of NEAR amplification of DNA from lysedspores.

Average AUC values from amplified product masses compared for lysed andunlysed samples. Lysed spore samples were then added to NEAR master mixand run for 10 minutes at 55° C., heat denatured for 4 minutes at 94°C., and run on the mass spec for analysis. AUC values of product peakswere averaged and compared (n=3).

FIG. 20. Demonstration of the capture and extension approach for surfacedetection of the NEAR assay.

A.) Average binding (NEAR positive reaction product with no addedpolymerase), B.) 500,000 target (NEAR positive reaction product withadded polymerase), and C.) No target (NEAR negative reaction with addedpolymerase) are compared. The NEAR assay was run for 10 minutes at 55°C., heat denatured at 94° C. for 4 minutes, then added to the plate withcapture probe bound to the surface on the 5′ end. Polymerase is added toone well of the positive reaction. The plate is incubated at 55° C. for30 min, washed, SYBR II added, washed 3 times, and read on a Tecan platereader (495 nm excitation/530 nm emission).

FIG. 21. Pseudo-real-time fluorescence detection of the NEAR FRET assaywith a single template immobilized on a surface in the presence(squares) and absence (open triangles) of 1E+6 copies of genomic DNA.

NEAR reaction was performed in flat bottom 96-well plates covered withneutravidin. Solution of 1 μM FRET-labeled reverse template wasincubated with gentle mixing for 1 hr at 37° C. Wells were washed 3times with a PBS-Tween solution to release unbound template. NEARreaction mix was added to the wells (one for each time point taken) andincubated at 58° C. on a heating block in a shaking incubator set to 135RPM. Time points were taken by adding 1 μL EDTA to the well to stop thereaction. The fluorescence was read from the bottom using a Tecan 100plate reader.

DETAILED DESCRIPTION

Provided herein are methods for the exponential amplification of shortDNA or RNA sequences.

Target nucleic acids of the present invention include double-strandedand single-stranded nucleic acid molecules. The nucleic acid may be, forexample, DNA or RNA. Where the target nucleic acid is an RNA molecule,the molecule may be, for example, double-stranded, single-stranded, orthe RNA molecule may comprise a target sequence that is single-stranded.Target nucleic acids include, for example, genomic, plasmid,mitochondrial, cellular, and viral nucleic acid. The target nucleic acidmay be, for example, genomic, chromosomal, plasmid DNA, a gene, any typeof cellular RNA, or a synthetic oligonucleotide. By “genomic nucleicacid” is meant any nucleic acid from any genome, for example, includinganimal, plant, insect, and bacterial genomes, including, for example,genomes present in spores. Target nucleic acids further includemicroRNAs and siRNAs.

MicroRNAs, miRNAs, or small temporal RNAs (stRNAs), are shortsingle-stranded RNA sequences, about 21-23 nucleotides long that areinvolved in gene regulation. MicroRNAs are thought to interfere with thetranslation of messenger RNAs as they are partially complementary tomessenger RNAs. (see, for example, Ruvkun, G I, Science 294:797-99(2001); Lagos-Quintana, M., et al., Science 294:854-58 (2001); Lau, N.C., et al, Science 294:858-62 (2001); Lee, R. C., and Ambros, V.,Science 294:862-64 (2001); Baulcombe, D., et al., Science 297:2002-03(2002); Llave, c., Science 297:2053-56 (2002); Hutvagner, G., andZamore, P. D., Science 297:2056-60 (2002)). MicroRNA may also have arole in the immune system, based on studies recently reported inknock-out mice. (see, for example, Wade, N., “Studies Reveal and ImmuneSystem Regulator” New York Times, Apr. 27, 2007). MicroRNA precursorsthat may also be detected using the methods of the present inventioninclude, for example, the primary transcript (pri-miRNA) and thepre-miRNA stem-loop-structured RNA that is further processed into miRNA.

Short interfering RNAs, or siRNAs are at least partiallydouble-stranded, about 20-25 nucleotide long RNA molecules that arefound to be involved in RNA interference, for example, in thedown-regulation of viral replication or gene expression (see for exampleZamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00101846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99107409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831).

The use of the term “target sequence” may refer to either the sense orantisense strand of the sequence, and also refers to the sequences asthey exist on target nucleic acids, amplified copies, or amplificationproducts, of the original target sequence. The amplification product maybe a larger molecule that comprises the target sequence, as well as atleast one other sequence, or other nucleotides. The length of the targetsequence, and the guanosine:cytosine (GC) concentration (percent), isdependent on the temperature at which the reaction is run; thistemperature is dependent on the stability of the polymerases and nickingenzymes used in the reaction. Those of ordinary skill in the art may runsample assays to determine the appropriate length and GC concentrationfor the reaction conditions. For example, where the polymerase andnicking enzyme are stable up to 60° C., then the target sequence may be,for example, from 19 to 50 nucleotides in length, or for example, from20 to 45, 20 to 40, 22-35, or 23 to 32 nucleotides in length. The GCconcentration under these conditions may be, for example, less than 60%,less than 55%, less than 50%, or less than 45%. The target sequenceshould not contain nicking sites for any nicking enzymes that will beincluded in the reaction mix.

The target sequences may be amplified in many types of samplesincluding, but not limited to samples containing spores, viruses, cells,nucleic acid from prokaryotes or eukaryotes, or any free nucleic acid.For example, the assay can detect the DNA on the outside of sporeswithout the need for lysis. The sample may be isolated from any materialsuspected of containing the target sequence. For example, for animals,for example, mammals, such as, for example, humans, the sample maycomprise blood, bone marrow, mucus, lymph, hard tissues, for example,liver, spleen, kidney, lung, or ovary, biopsies, sputum, saliva, tears,feces, or urine. Or, the target sequence may be present in air, plant,soil, or other materials suspected of containing biological organisms.

Target sequences may be present in samples that may also containenvironmental and contaminants such as dust, pollen, and diesel exhaust,or clinically relevant matrices such as urine, mucus, or saliva. Targetsequences may also be present in waste water, drinking water, air, milk,or other food. Depending on the concentration of these contaminants,sample purification methods known to those of ordinary skill in the artmay be required to remove inhibitors for successful amplification.Purification may, for example, involve the use of detergent lysates,sonication, vortexing with glass beads, or a French press. Thispurification could also result in concentration of the sample target.Samples may also, for be further purified, for example, by filtration,phenol extraction, chromatography, ion exchange, gel electrophoresis, ordensity dependent centrifugation. The sample can be added directly tothe reaction mix or pre-diluted and then added.

An oligonucleotide is a molecule comprising two or moredeoxyribonucleotides or ribonucleotides, for example, more than three.The length of an oligonucleotide will depend on how it is to be used.The oligonucleotide may be derived synthetically or by cloning.

The term “complementary” as it refers to two nucleic acid sequencesgenerally refers to the ability of the two sequences to form sufficienthydrogen bonding between the two nucleic acids to stabilize adouble-stranded nucleotide sequence formed by hybridization of the twonucleic acids.

As used herein, “hybridization” and “binding” are used interchangeablyand refer to the non-covalent binding or “base pairing” of complementarynucleic acid sequences to one another. Whether or not a particular proberemains base paired with a polynucleotide sequence depends on the degreeof complementarity, the length of the probe, and the stringency of thebinding conditions. The higher the stringency, the higher must be thedegree of complementarity, and/or the longer the probe for binding orbase pairing to remain stable.

As used herein, “stringency” refers to the combination of conditions towhich nucleic acids are subjected that cause double-stranded nucleicacid to dissociate into component single strands such as pH extremes,high temperature, and salt concentration. The phrase “high stringency”refers to hybridization conditions that are sufficiently stringent orrestrictive such that only specific base pairings will occur. Thespecificity should be sufficient to allow for the detection of uniquesequences using an oligonucleotide probe or closely related sequenceunder standard Southern hybridization protocols (as described in J. Mol.Biol. 98:503 (1975)).

Templates are defined as oligonucleotides that bind to a recognitionregion of the target and also contain a nicking enzyme binding regionupstream of the recognition region and a stabilizing region upstream tothe nicking enzyme binding region.

By “recognition region” is meant a nucleic acid sequence on the templatethat is complementary to a nucleic acid sequence on the target sequence.By “recognition region on the target sequence” is meant the nucleotidesequence on the target sequence that is complementary to, and binds to,the template.

By “stabilizing region” is meant a nucleic acid sequence having, forexample, about 50% GC content, designed to stabilize the molecule for,for example, the nicking and/or extension reactions.

In describing the positioning of certain sequences on nucleic acidmolecules, such as, for example, in the target sequence, or thetemplate, it is understood by those of ordinary skill in the art thatthe terms “3′” and “5′” refer to a location of a particular sequence orregion in relation to another. Thus, when a sequence or a region is 3′to or 3′ of another sequence or region, the location is between thatsequence or region and the 3′ hydroxyl of that strand of nucleic acid.When a location in a nucleic acid is 5′ to or 5′ of another sequence orregion, that means that the location is between that sequence or regionand the 5′ phosphate of that strand of nucleic acid.

The polymerase is a protein able to catalyze the specific incorporationof nucleotides to extend a 3′ hydroxyl terminus of a primer molecule,such as, for example, the template oligonucleotide, against a nucleicacid target sequence. The polymerase may be, for example, thermophilicso that it is active at an elevated reaction temperature. It may also,for example, have strand displacement capabilities. It does not,however, need to be very processive (30-40 nucleotides for a singlesynthesis is sufficient). If the polymerase also has reversetranscription capabilities (such as Bst (large fragment), 9° N,Therminator, Therminator II, etc.) the reaction can also amplify RNAtargets in a single step without the use of a separate reversetranscriptase. More than one polymerase may be included in the reaction,in one example one of the polymerases may have reverse transcriptaseactivity and the other polymerase may lack reverse transcriptaseactivity. The polymerase may be selected from, for example, the groupconsisting of one or more of the polymerases listed in Table 1.

TABLE 1 Polymerase Bst DNA polymerase Bst DNA polymerase (Largefragment) 9°Nm DNA polymerase Phi29 DNA polymerase DNA polymerase I (E.coli) DNA polymerase I, Large (Klenow) fragment Klenow fragment (3′-5′exo-) T4 DNA polymerase T7 DNA polymerase Deep VentR ™ (exo-) DNAPolymerase Deep VentR ™DNA Polymerase DyNAzyme ™ EXT DNA DyNAzyme ™ IIHot Start DNA Polymerase Phusion ™ High-Fidelity DNA PolymeraseTherminator ™ DNA Polymerase Therminator ™ II DNA Polymerase VentR ®DNAPolymerase VentR ® (exo-) DNA Polymerase RepliPHFM Phi29 DNA PolymeraserBst DNA Polymerase, Large Fragment (IsoTherm ™ DNA Polymerase)MasterAmpTM AmpliThermTM DNA Polymerase Tag DNA polymerase Tth DNApolymerase Tfl DNA polymerase Tgo DNA polymerase SP6 DNA polymerase TbrDNA polymerase DNA polymerase Beta ThermoPhi DNA polymerase

“Nicking” refers to the cleavage of only one strand of thedouble-stranded portion of a fully or partially double-stranded nucleicacid. The position where the nucleic acid is nicked is referred to asthe nicking site or nicking enzyme site. The recognition sequence thatthe nicking enzyme recognizes is referred to as the nicking enzymebinding site. “Capable of nicking” refers to an enzymatic capability ofa nicking enzyme.

The nicking enzyme is a protein that binds to double-stranded DNA andcleaves one strand of a double-stranded duplex. The nicking enzyme maycleave either upstream or downstream of the binding site, or nickingenzyme recognition site. In exemplary embodiments, the reactioncomprises the use of nicking enzymes that cleave or nick downstream ofthe binding site (top strand nicking enzymes) so that the productsequence does not contain the nicking site. Using an enzyme that cleavesdownstream of the binding site allows the polymerase to more easilyextend without having to displace the nicking enzyme. The nicking enzymemust be functional in the same reaction conditions as the polymerase, sooptimization between the two ideal conditions for both is necessary.Nicking enzymes are available from, for example, New England Biolabs(NEB) and Fermentas. The nicking enzyme may, for example, be selectedfrom the group consisting of one or more of the nicking enzymes listedin Table 2.

TABLE 2 Alternate Nicking Enzyme Name Nb.BbvCI Nb.Bpu101 Nb.Bsal Nb.BsmlNb.BsrDI Nb.BstNBIP Nb.BstSEIP Nb.Btsl Nb.Sapl Nt.Alwl Nt.BbvCINt.BhaIIiP Nt.Bpu101 Nt.Bpu10IB Nt.Bsal Nt.BsmAI Nt.BsmBI Nt.BspD61Nt.BspQI Nt.Bst91 Nt.BstNBI N.BstNB I Nt.BstSEI Nt.CviARORFMPNt.CviFRORFAP Nt.CviPIl Nt.CviPlim Nt.CviQII Nt.CviQXI Nt.EsaSS1198PNt.Mlyl Nt.Sapl

Nicking enzymes may be, for example, selected from the group consistingof Nt.BspQI(NEB), Nb.BbvCI(NEB), Nb.BsmI(NEB), Nb.BsrDI(NEB),Nb.BtsI(NEB), Nt.AlwI(NEB), Nt.BbvCI(NEB), Nt.BstNBI(NEB),Nt.CviPII(NEB), Nb.BpulOl(Fermantas), and Nt.BpulOl(Fermentas). Incertain embodiments, the nicking enzyme is selected from the groupconsisting of Nt.NBst.NBI, Nb.BsmI, and Nb.BsrDI. Those of ordinaryskill in the art are aware that various nicking enzymes other than thosementioned specifically herein may be used in the methods of the presentinvention.

Nicking enzymes and polymerases of the present invention may be, forexample, stable at room temperature, the enzymes may also, for example,be stable at temperatures up to 37° C., 42° C., 60° C., 65° C., 70° C.,75° C., 80° C., or 85° C. In certain embodiments, the enzymes are stableup to 60° C.

Product or amplified product is defined as the end result of theextension of the template along the target that is nicked, released, andthen feeds back into the amplification cycle as a target for theopposite template.

A “native nucleotide” refers to adenylic acid, guanylic acid, cytidylicacid, thymidylic acid, or uridylic acid. A “derivatized nucleotide” is anucleotide other than a native nucleotide.

The reaction may be conducted in the presence of native nucleotides,such as, for example, dideoxyribonucleoside triphosphates (dNTPs). Thereaction may also be carried out in the presence of labeled dNTPs, suchas, for example, radiolabels such as, for example, ³²P, ³³P, ¹²⁵I, or³⁵S, enzyme labels such as alkaline phosphatase, fluorescent labels suchas fluorescein isothiocyanate (FITC), biotin, avidin, digoxigenin,antigens, haptens, or fluorochromes. These derivatized nucleotides may,for example, be present in the templates.

By “constant temperature,” “isothermal conditions” or “isothermally” ismeant a set of reaction conditions where the temperature of the reactionis kept essentially constant during the course of the amplificationreaction. An advantage of the amplification method of the presentinvention is that the temperature does not need to be cycled between anupper temperature and a lower temperature. The nicking and the extensionreaction will work at the same temperature or within the same narrowtemperature range. However, it is not necessary that the temperature bemaintained at precisely one temperature. If the equipment used tomaintain an elevated temperature allows the temperature of the reactionmixture to vary by a few degrees, this is not detrimental to theamplification reaction, and may still be considered to be an isothermalreaction.

The term “multiplex amplification” refers to the amplification of morethan one nucleic acid of interest. For example, it can refer to theamplification of multiple sequences from the same sample or theamplification of one of several sequences in a sample as discussed, forexample, in U.S. Pat. Nos. 5,422,252; and 5,470,723, which provideexamples of multiplex strand displacement amplification. The term alsorefers to the amplification of one or more sequences present in multiplesamples either simultaneously or in step-wise fashion.

Template Design

Forward and Reverse templates are designed so that there is astabilizing region at the 5′ end, a nicking site downstream of thestabilizing region, and a recognition region downstream of the nickingsite on the 3′ end of the oligonucleotide. The total oligo length canrange from 19 to 40, for example from 19-40, 23-40, 20-24, 23-24, 23-32,25-40, 27-40, or 27-35 nucleotides depending on the length of eachindividual region, the temperature, the length of the target sequence,and the GC concentration. The templates may be designed so that they,together, would bind to less than or equal to 100% of the targetsequence, one binding to the sense strand, and one to the antisensestrand. For example, where the forward template binds to about 60% ofthe target antisense strand, the reverse template may, for example, bindto about 40% of the target sense strand. The templates may be designedto allow for spacer bases on the target sequence, that do not bind toeither template. The templates thus may be designed to bind to about30%, about 40%, about 50%, or about 60% of the target sequence.

The recognition region of the forward template is designed to beidentical to the 5′ region of the target sense strand and complementaryto the 3′ end of the target site antisense strand, for example, 8-16,9-16, 10-16, 10-15, or 11-14 nucleotides long. In exemplary embodiments,the length is 12-13 nucleotides. The recognition region of the reversetemplate is designed to be complementary to the 3′ end of the targetsite sense strand, for example, 8-16, 9-16, 10-16, 1015, or 11-14nucleotides long. In exemplary embodiments, the length is 12-13nucleotides.

In certain embodiments, the lengths of the recognition regions areadjusted so that there is at least one nucleotide in the target sequencethat is not in the forward template's recognition region and also doesnot have its complement in the reverse template's recognition region.These spacer bases are nucleotides contained within the target sequencethat lie in between the 3′ ends of the forward and reverse templates. Incertain embodiments, 5 spacer bases or less are present in the targetsequence. In exemplary embodiments, the number of spacer bases is 2 to3. In certain embodiments, the number of spacer bases is 1, 2, 3, 4, or5. These spacer bases allow for distinction of the true amplifiedproduct from any background products amplified by extension due tooverlapping templates in a similar manner to primer-dimers. Thisconsideration allows for improved discrimination between background andamplification of true target. However, these spacer bases are notrequired for the amplification to proceed.

The nicking site sequence of the template depends on which nickingenzyme is chosen for each template. Different nicking enzymes may beused in a single assay, but a simple amplification may, for example,employ a single nicking enzyme for use with both templates. Thus, theembodiments of the present invention include those where both templatescomprise recognition sites for the same nicking enzyme, and only onenicking enzyme is used in the reaction. In these embodiments, both thefirst and second nicking enzymes are the same. The present inventionalso includes those embodiments where each template comprises arecognition site for a different nicking enzyme, and two nicking enzymesare used in the reaction.

For example, in the case of Nt.BstNBI, the enzyme binding site is5′-GAGTC-3′ and the enzyme nicks the top strand four nucleotides downstream of this site (i.e., GAGTCNNNNA). The amplification reaction showslittle dependence on the sequence of these four nucleotides (N), thoughoptimal sequence of this region is 25% or less GC content and with athymine adjacent to the 5′ nucleotide of the binding region. The latterstipulation allows for the priming ability of products that have anadditional adenine added on by the polymerase. The sequence of the fournucleotides can be optimized to create or eliminate the presence ofhairpins, self-dimers, or heterodimers, depending on the application.

The stabilizing region on the 5′ end of the template oligonucleotide isdesigned to be roughly 50% GC. Thus, the GC content may be, for example,about 40%-60%, about 42%-58%, about 44%-56%, about 46%-54%, about48%-52%, or about 49%-51%. These parameters result in a stabilizingregion length of 8-11 nucleotides for the Nt.BstNBI enzyme, thoughlengths as short as 6 and as long as 15 nucleotides have been tested andwere shown to work in this amplification method. Longer stabilizingregions or increased % GC to greater than 50% could further stabilizethe nicking and extension reactions at higher reaction temperatures. Thesequence of the 5′ stabilizing regions of forward and reverse templatesare usually identical, but can be varied if the aim is to capture eachproduct strand independently. The sequence of this region should notinterfere with the nicking site or the recognition region, though shortinternal hairpins within the template sequence have been shown to haveimproved real-time results.

The templates of the present invention may include, for example,spacers, blocking groups, and modified nucleotides. Modified nucleotidesare nucleotides or nucleotide triphosphates that differ in compositionand/or structure from natural nucleotide and nucleotide triphosphates.Modified nucleotide or nucleotide triphosphates used herein may, forexample, be modified in such a way that, when the modifications arepresent on one strand of a double-stranded nucleic acid where there is arestriction endonuclease recognition site, the modified nucleotide ornucleotide triphosphates protect the modified strand against cleavage byrestriction enzymes. Thus, the presence of the modified nucleotides ornucleotide triphosphates encourages the nicking rather than the cleavageof the double-stranded nucleic acid. Blocking groups are chemicalmoieties that can be added to the template to inhibit targetsequence-independent nucleic acid polymerization by the polymerase.Blocking groups are usually located at the 3′ end of the template.Examples of blocking groups include, for example, alkyl groups,non-nucleotide linkers, phosphorothioate, alkane-diol residues, peptidenucleic acid, and nucleotide derivatives lacking a 3′-OH, including, forexample, cordycepin. Examples of spacers, include, for example, C3spacers. Spacers may be used, for example, within the template, andalso, for example, at the 5′ end, to attach other groups, such as, forexample, labels.

Detailed Mechanism of Amplification

NEAR amplification requires the presence of a nucleic acid target, atleast two template oligonucleotides, a thermophilic nicking enzyme, athermophilic polymerase, and buffer components all held at the reactiontemperature. The recognition region of the templates interacts with thecomplementary target sequence. Since the melting temperature of thecomplementary regions of the target and template is well below thereaction temperature, the interaction between the two nucleic acidstrands is transient, but allows enough time for a thermophilicpolymerase to extend from the 3′ end of the template along the targetstrand. Experiments have shown that certain polymerases bind tosingle-stranded oligonucleotides. The pre-formation of this complex canfacilitate the speed of the amplification process.

For a double-stranded target, both templates can interact with thecorresponding target strands simultaneously (forward template with theantisense strand and reverse template with the sense strand) during thenormal breathing of double-stranded DNA. The target may also begenerated by a single or double nick sites within the genome sequence.For a single-stranded target (either RNA or DNA), the reverse templatebinds and extends first (FIG. 1, Step 1 and 2). The extended sequencecontains the complement to the forward template. The forward templatethen displaces a region of the target and binds to the 3′ synthesizedregion complementary to the recognition region of the forward template(Step 3). Alternatively, another reverse template can also displace theinitial extended reverse template at the recognition region to create asingle-stranded extended reverse template for the forward template tobind. The initial binding and extension of the templates is facilitatedby a non-processive polymerase that extends shorter strands of DNA sothat the melting temperature of the synthesized product is at or nearthe reaction temperature; therefore, a percentage of the product becomessingle-stranded once the polymerase dissociates. The single-strandedproduct is then available for the next template recognition site to bindand polymerase to extend.

The forward template is extended to the 5′ end of the reverse template,creating a double-stranded nicking enzyme binding site for the reversetemplate (Step 5). The nicking enzyme then binds to the duplex and nicksdirectly upstream of the recognition sequence of the reverse templatestrand (in the case of a top-strand nicking enzyme) (Step 6). Thenucleic acid sequence downstream of the nick is either released (if themelting temperature is near the reaction temperature) and/or isdisplaced by the polymerase synthesis from the 3-OH nick site.

Polymerase extends along the forward template to the 5′ end of theforward template (Step 8).

The double-strand formed from the extension of both templates creates anicking enzyme binding site on either end of the duplex. Thisdouble-strand is termed the NEAR amplification duplex. When nickingenzyme binds and nicks, either the target product located in between thetwo nick sites (with 5′-phosphate and 3-OH) is released, usually rangingin length from (but is not limited to) 23 to 29 bases (Steps 9-11A), orthe singly-nicked product containing the target product and the reversecomplement of the nick site and stability region of the template(usually 36 to 48 bases in length) is released (Steps 9-11B). The ratioof products 1 to 2 can be adjusted by varying the concentrations of thetemplates. The forward:reverse template ratio may vary from, forexample, molar ratios of 100:1, 75:1; 50:1, 40:1, 30:1, 20:1, 10:1, 5:1,2.5:1, 1:1, 1:2.5, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:75, or 1:100.The ratio of products (A to B) is dependent on the ratio of nickingenzyme to polymerase, i.e. a higher concentration of polymerase resultsin more of the longer length product (B) since there is comparativelyless nicking enzyme to nick both strands simultaneously before thepolymerase extends. Since displaced/released product of the reversetemplate feeds into the forward template and vice versa, exponentialamplification is achieved. The nicking enzyme:polymerase ratio may varyfrom, for example, enzyme unit ratios of 20:1, 15:1; 10:1, 5:1, 4:1,3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20. Incertain embodiments, the ratio of nicking enzyme to polymerase may, forexample, be 1:3, 1:2, 1:1.5, or 1:0.8. Those of ordinary skill in theart recognize that these ratios may represent rounded values. Thisnicking and polymerase extension process continues until one of theresources (usually dNTPs or enzyme) is exhausted.

The time that the reaction is run may vary from, for example, 1-20minutes, or 1-10, 1-8, 1-5, 1-2.5, 2.5-5, 2.5-8, 2.5-10, or 2.5-20minutes.

The methods of the present invention do not require the use oftemperature cycling, as often is required in methods of amplification todissociate the target sequence from the amplified nucleic acid. Thetemperature of the reaction may vary based on the length of thesequence, and the GC concentration, but, as understood by those ofordinary skill in the art, the temperature should be high enough tominimize non-specific binding. The temperature should also be suitablefor the enzymes of the reaction, the nicking enzyme and the polymerase.For example, the reaction may be run at 37° C.-85° C., 37° C.-60° C.,54° C.-60° C., and, in exemplary embodiments, from 55° C.-59° C.

The polymerase may be mixed with the target nucleic acid moleculebefore, after, or at 15 the same time as, the nicking enzyme. Inexemplary embodiments, a reaction buffer is optimized to be suitable forboth the nicking enzyme and the polymerase.

Reactions may be allowed to completion, that is, when one of theresources is exhausted. Or, the reaction may be stopped using methodsknown to those of ordinary skill in the art, such as, for example, heatdenaturation, or the addition of EDTA, high salts, or detergents. Inexemplary embodiments, where mass spectrometry is to be used followingamplification, EDTA may be used to stop the reaction.

Reaction Components

In a 1.5 mL Eppendorf tube combine the following reagents in order fromtop to bottom:

μL Per Reagent Added: Reaction H₂O 31.4 10X Thermopol Buffer (NEB) 5 10XNEB Buffer 3 2.5 100 mM MgSO₄ 4.5 10 mM dNTPs 1.5 8 U/μl Bst Pol 0.6 10U/μl N.BstNBI 1.5 20 μM Forward Template 0.25 20 μM Reverse Template0.25 Total reaction mixture 47.5 Target sample 2.5 Total Reaction Volume50 μL

The concentrations of components for the reaction conditions in thisexample are as follows:

Concentration Component 45.7 mM Tris-HGI 13.9 mM KCl 10 mM (NH₄)₂SO₂ 50mM NaCl 0.5 mM DTT 15 mM MgCl₂ 0.10% Triton X-100 0.008 mM EDTA 6 μg/mLBSA 3.90% Glycerol (can be lower if using more concentrated enzymestock) 0.3 U/μL NT.BstNBI 0.1-0.4 U/μL Bst polymerase (large fragment)0.1 μM Forward template 0.1 μM Reverse template

Variations in buffer conditions, MgSO₄ concentration, polymeraseconcentration, and template concentrations all can be optimized based onthe assay sequence and desired detection method. The amount of glycerolmay, for example, be lowered if a more concentrated enzyme stock isused. Also, those of ordinary skill in the art recognize that thereaction may be run without EDTA or BSA; these components may be presentin the reaction as part of the storage buffers for the enzymes. Thevolumes can be scaled for larger or smaller total reaction volumes. Thevolume is usually between 50 μL and 100 μL.

The template concentrations are typically in excess of the concentrationof target. The concentrations of the forward and reverse templates canbe at the same or at different concentrations to bias the amplificationof one product over the other. The concentration of each is usuallybetween 10 nM and 1 μM.

Additives such as BSA, non-ionic detergents such as Triton X-100 orTween-20, DMSO, DTT, and RNase inhibitor may be included foroptimization purposes without adversely affecting the amplificationreaction.

Preparing/Adding Target

Targets may be diluted in 1× Thermopol Buffer II, 1×TE (pH 7.5) or H₂O.Hot start conditions allow for faster, more specific amplification. Inthis case, the reaction mix (minus either enzymes or templates andtarget) is heated to the reaction temperature for 2 minutes, after whichthe reaction mix is added to the other component (enzymes ortemplates/target). The target can be added in any volume up to the totalamount of water required in the reaction. In this case, the target wouldbe diluted in water. In the example above for a 500 μL total reactionvolume, 2.5 μL of the prepared target should be added per reaction tobring the total reaction volume to 50 μL.

Running the Reaction

The reaction is run at a constant temperature, usually between 54° C.and 60° C. for the enzyme combination of Bst polymerase (large fragment)and Nt.Bst.NB 1 nicking enzyme. Other enzyme combinations may be usedand the optimal reaction temperature will be based on the optimaltemperature for both the nicking enzyme and polymerase to work inconcert as well as the melting temperature of the reaction products. Thereaction is held at temperature for 2.5 to 10 minutes until the desiredamount of amplification is achieved. The reaction may be stopped byeither a heat denaturation step to denature the enzymes (when usingenzymes that can be heat-killed). Alternatively, the reaction may bestopped by adding EDTA to the reaction.

Readout

The amplified target sequence may be detected by any method known to oneof ordinary skill in the art. By way of non-limiting example, several ofthese known methods are presented herein. In one method, amplifiedproducts may be detected by gel electrophoresis, thus detecting reactionproducts having a specific length. The nucleotides may, for example, belabeled, such as, for example, with biotin. Biotin-labeled amplifiedsequences may be captured using avidin bound to a signal generatingenzyme, for example, peroxidase.

Nucleic acid detection methods may employ the use of dyes thatspecifically stain double-stranded DNA. Intercalating dyes that exhibitenhanced fluorescence upon binding to DNA or RNA are a basic tool inmolecular and cell biology. Dyes may be, for example, DNA or RNAintercalating fluorophores and may include but are not limited to thefollowing examples:

Acridine orange, ethidium bromide, Hoechst dyes, PicoGreen, propidiumiodide, SYBR I (an asymmetrical cyanine dye), SYBR II, TOTO (a thiaxoleorange dimer) and YOYO (an oxazole yellow dimer). Dyes provide anopportunity for increasing the sensitivity of nucleic acid detectionwhen used in conjunction with various detection methods and may havevarying optimal usage parameters. For example ethidium bromide iscommonly used to stain DNA in agarose gels after gel electrophoresis andduring peR (Hiquchi et al., Nature Biotechnology 10; 413-417, April1992), propidium iodide and Hoechst 33258 are used in flow cytometry todetermine DNA ploidy of cells, SYBR Green 1 has been used in theanalysis of double-stranded DNA by capillary electrophoresis with laserinduced fluorescence detection and Pico Green has been used to enhancethe detection of double-stranded DNA after matched ion pairpolynucleotide chromatography (Singer et al., Analytical Biochemistry249,229-238 1997).

Nucleic acid detection methods may also employ the use of labelednucleotides incorporated directly into the target sequence or intoprobes containing complementary sequences to the target of interested.Such labels may be radioactive and/or fluorescent in nature and can beresolved in any of the manners discussed herein.

Methods of detecting and/or continuously monitoring the amplification ofnucleic acid products are also well known to those skilled in the artand several examples are described below.

The production or presence of target nucleic acids and nucleic acidsequences may be detected and monitored by Molecular Beacons. MolecularBeacons are hair-pin shaped oligonucleotides containing a fluorophore onone end and a quenching dye on the opposite end. The loop of thehair-pin contains a probe sequence that is complementary to a targetsequence and the stem is formed by annealing of complementary armsequences located on either side of the probe sequence. A fluorophoreand a quenching molecule are covalently linked at opposite ends of eacharm. Under conditions that prevent the oligonucleotides from hybridizingto its complementary target or when the molecular beacon is free insolution the fluorescent and quenching molecules are proximal to oneanother preventing fluorescence resonance energy transfer (FRET). Whenthe molecular beacon encounters a target molecule, hybridization occurs;the loop structure is converted to a stable more rigid conformationcausing separation of the fluorophore and quencher molecules leading tofluorescence (Tyagi et al. Nature Biotechnology 14: March 1996,303-308). Due to the specificity of the probe, the generation offluorescence is exclusively due to the synthesis of the intendedamplified product.

Molecular beacons are extraordinarily specific and can discern a singlenucleotide polymorphism. Molecular beacons can also be synthesized withdifferent colored fluorophores and different target sequences, enablingseveral products in the same reaction to be quantitated simultaneously.For quantitative amplification processes, molecular beacons canspecifically bind to the amplified target following each cycle ofamplification, and because non-hybridized molecular beacons are dark, itis not necessary to isolate the probe-target hybrids to quantitativelydetermine the amount of amplified product. The resulting signal isproportional to the amount of amplified product. This can be done inreal time. As with other real time formats, the specific reactionconditions must be optimized for each primer/probe set to ensureaccuracy and precision.

The production or presence of target nucleic acids and nucleic acidsequences may also be detected and monitored by Fluorescence resonanceenergy transfer (FRET). FRET is an energy transfer mechanism between twochromophores: a donor and an acceptor molecule. Briefly, a donorfluorophore molecule is excited at a specific excitation wavelength. Thesubsequent emission from the donor molecule as it returns to its groundstate may transfer excitation energy to the acceptor molecule through along range dipole-dipole interaction. The intensity of the emission ofthe acceptor molecule can be monitored and is a function of the distancebetween the donor and the acceptor, the overlap of the donor emissionspectrum and the acceptor absorption spectrum and the orientation of thedonor emission dipole moment and the acceptor absorption dipole moment.FRET is a useful tool to quantify molecular dynamics, for example, inDNA-DNA interactions as seen with Molecular Beacons. For monitoring theproduction of a specific product a probe can be labeled with a donormolecule on one end and an acceptor molecule on the other. Probe-targethybridization brings a change in the distance or orientation of thedonor and acceptor and FRET change is observed. (Joseph R. Lakowicz,“Principles of Fluorescence Spectroscopy”, Plenum PublishingCorporation, 2nd edition (Jul. 1, 1999)).

The production or presence of target nucleic acids and nucleic acidsequences may also be detected and monitored by Mass Spectrometry. MassSpectrometry is an analytical technique that may be used to determinethe structure and quantity of the target nucleic acid species and can beused to provide rapid analysis of complex mixtures. Following themethod, samples are ionized, the resulting ions separated in electricand/or magnetic fields according to their mass-to-charge ratio, and adetector measures the mass-to-charge ratio of ions. (Crain, P. F. andMcCloskey, J. A., Current Opinion in Biotechnology 9: 25-34 (1998)).Mass spectrometry methods include, for example, MALDI, MALDIITOF, orElectrospray. These methods may be combined with gas chromatography(GC/MS) and liquid chromatography (LC/MS). MS has been applied to thesequence determination of DNA and RNA oligonucleotides (Limbach P.,MassSpectrom. Rev. 15: 297-336 (1996); Murray K., J. Mass Spectrom. 31:1203-1215 (1996)). MS and more particularly, matrix-assisted laserdesorption/ionization MS (MALDI MS) has the potential of very highthroughput due to high-speed signal acquisition and automated analysisoff solid surfaces. It has been pointed out that MS, in addition tosaving time, measures an intrinsic property of the molecules, andtherefore yields a significantly more informative signal (Koster H. etal., Nature Biotechnol., 14: 1123-1128 (1996)).

The production or presence of target nucleic acids and nucleic acidsequences may also be detected and monitored by various methods of gelelectrophoresis. Gel electrophoresis involves the separation of nucleicacids through a matrix, generally a cross-linked polymer, using anelectromotive force that pulls the molecules through the matrix.Molecules move through the matrix at different rates causing aseparation between products that can be visualized and interpreted viaanyone of a number of methods including but not limited to;autoradiography, phosphorimaging, and staining with nucleic acidchelating dyes.

The production or presence of target nucleic acids and nucleic acidsequences may also be detected and monitored by capillary gelelectrophoresis. Capillary-gel Electrophoresis (CGE) is a combination oftraditional gel electrophoresis and liquid chromatography that employs amedium such as polyacrylamide in a narrow bore capillary to generatefast, high-efficient separations of nucleic acid molecules with up tosingle base resolution. CGE is commonly combined with laser inducedfluorescence (LIF) detection where as few as six molecules of stainedDNA can be detected. CGE/LIF detection generally involves the use offluorescent DNA intercalating dyes including ethidium bromide, YOYO andSYBR Green 1 but can also involve the use of fluorescent DNA derivativeswhere the fluorescent dye is covalently bound to the DNA. Simultaneousidentification of several different target sequences can be made usingthis method.

The production or presence of target nucleic acids and nucleic acidsequences may also be detected and monitored by various surface capturemethods. This is accomplished by the immobilization of specificoligonucleotides to a surface producing a biosensor that is both highlysensitive and selective. Surfaces used in this method may include butare not limited to gold and carbon and may use a number of covalent ornoncovalent coupling methods to attach the probe to the surface. Thesubsequent detection of a target DNA can be monitored by a variety ofmethods.

Electrochemical methods generally involve measuring the cathodic peak ofintercalators, such as methylene blue, on the DNA probe electrode andvisualized with square wave voltammograms. Binding of the targetsequence can be observed by a decrease in the magnitude of thevoltammetric reduction signals of methylene blue as it interacts withdsDNA and ssDNA differently reflecting the extent of the hybridformation.

Surface Plasmon Resonance (SPR) can also be used to monitor the kineticsof probe attachment as well as the process of target capture. SPR doesnot require the use of fluorescence probes or other labels. SPR relieson the principle of light being reflected and refracted on an interfaceof two transparent media of different refractive indexes. Usingmonochromatic and p-polarized light and two transparent media with aninterface comprising a thin layer of gold, total reflection of light isobserved beyond a critical angle, however the electromagnetic fieldcomponent of the light penetrates into the medium of lower refractiveindex creating an evanescent wave and a sharp shadow (surface plasmonresonance). This is due to the resonance energy transfer between thewave and the surface plasmons. The resonance conditions are influencedby the material absorbed on the thin metal film and nucleic acidmolecules, proteins and sugars concentrations are able to be measuredbased on the relation between resonance units and mass concentration.

The production or presence of target nucleic acids and nucleic acidsequences may also be detected and monitored by lateral flow devices.Lateral Flow devices are well known. These devices generally include asolid phase fluid permeable flow path through which fluid flows throughby capillary force. Examples include, but are not limited to, dipstickassays and thin layer chromatographic plates with various appropriatecoatings. Immobilized on the flow path are various binding reagents forthe sample, binding partners or conjugates involving binding partnersfor the sample and signal producing systems. Detection of samples can beachieved in several manners; enzymatic detection, nanoparticledetection, colorimetric detection, and fluorescence detection, forexample. Enzymatic detection may involve enzyme-labeled probes that arehybridized to complementary nucleic aid targets on the surface of thelateral flow device. The resulting complex can be treated withappropriate markers to develop a readable signal. Nanoparticle detectioninvolves bead technology that may use colloidal gold, latex andparamagnetic nanoparticles. In one example, beads may be conjugated toan anti-biotin antibody. Target sequences may be directly biotinylated,or target sequences may be hybridized to a sequence specificbiotinylated probes. Gold and latex give rise to colorimetric signalsvisible to the naked eye and paramagnetic particles give rise to anon-visual signal when excited in a magnetic field and can beinterpreted by a specialized reader.

Fluorescence-based lateral flow detection methods are also known, forexample, dual fluorescein and biotin-labeled oligo probe methods, UPT-NALP utilizing up-converting phosphor reporters composed of lanthanideelements embedded in a crystal (Corstjens et al., Clinical Chemistry,47:10, 1885-1893, 2001), as well as the use of quantum dots.

Nucleic acids can also be captured on lateral flow devices. Means ofcapture may include antibody dependent and antibody independent methods.Antibody-dependent capture generally comprises an antibody capture lineand a labeled probe of complementary sequence to the target.Antibody-independent capture generally uses non-covalent interactionsbetween two binding partners, for example, the high affinity andirreversible linkage between a biotinylated probe and a streptavidinline. Capture probes may be immobilized directly on lateral flowmembranes. Both antibody dependent and antibody independent methods maybe used in multiplexing.

The production or presence of target nucleic acids and nucleic acidsequences may also be detected and monitored by multiplex DNAsequencing. Multiplex DNA sequencing is a means of identifying targetDNA sequences from a pool of DNA. The technique allows for thesimultaneous processing of many sequencing templates. Pooled multipletemplates can be resolved into individual sequences at the completion ofprocessing. Briefly, DNA molecules are pooled, amplified and chemicallyfragmented. Products are fractionated by size on sequencing gels andtransferred to nylon membranes. The membranes are probed andautoradiographed using methods similar to those used in standard DNAsequencing techniques (Church et al., Science 1998 Apr. 8;240(4849):185-188). Autoradiographs can be evaluated and the presence oftarget nucleic acid sequence can be quantitated.

Kits

Kits of the present invention may comprise, for example, one or morepolymerases, forward and reverse templates, and one or more nickingenzymes, as described herein. Where one target is to be amplified, oneor two nicking enzymes may be included in the kit. Where multiple targetsequences are to be amplified, and the templates designed for thosetarget sequences comprise the nicking enzyme sites for the same nickingenzyme, then one or two nicking enzymes may be included. Or, where thetemplates are recognized by different nicking enzymes, more nickingenzymes may be included in the kit, such as, for example, 3 or more.

The kits of the present invention may also comprise one or more of thecomponents in any number of separate containers, packets, tubes, vials,microtiter plates and the like, or the components may be combined invarious combinations in such containers.

The components of the kit may, for example, be present in one or morecontainers, for example, all of the components may be in one container,or, for example, the enzymes may be in a separate container from thetemplates. The components may, for example, be lyophilized, freezedried, or in a stable buffer. In one example, the polymerase and nickingenzymes are in lyophilized form in a single container, and the templatesare either lyophilized, freeze dried, or in buffer, in a differentcontainer. Or, in another example, the polymerase, nicking enzymes, andthe templates are, in lyophilized form, in a single container. Or, thepolymerase and the nicking enzyme may be separated into differentcontainers.

Kits may further comprise, for example, dNTPs used in the reaction, ormodified nucleotides, cuvettes or other containers used for thereaction, or a vial of water or buffer for re-hydrating lyophilizedcomponents. The buffer used may, for example, be appropriate for bothpolymerase and nicking enzyme activity.

The kits of the present invention may also comprise instructions forperforming one or more methods described herein and/or a description ofone or more compositions or reagents described herein. Instructionsand/or descriptions may be in printed form and may be included in a kitinsert. A kit also may include a written description of an Internetlocation that provides such instructions or descriptions.

Kits may further comprise reagents used for detection methods, such as,for example, reagents used for FRET, lateral flow devices, dipsticks,fluorescent dye, colloidal gold particles, latex particles, a molecularbeacon, or polystyrene beads.

EXAMPLES Example 1 Detection of DNA NEAR Assay Products by GelElectrophoresis

The NEAR amplification reaction products can be visualized by gelelectrophoresis. In the absence of target, the templates (withcomplementary 3′ bases) overlap by one or more bases, polymerase extendsin each direction to generate the NEAR amplification duplex (FIG. 1B);and the amplification proceeds in a similar mechanism to the NEARamplification to amplify a product that is two bases shorter than thetarget amplified product. In the case of a 25mer assay where thetemplates end in A and T, the resulting background product is 23 bases.The 27mer assay also forms a 23mer background and 27mer product. Longerreaction products are also amplified. The sequence of these products ishypothesized to be due to the polymerase extension before the nickingenzyme can nick both sides of the NEAR amplification duplex, accordingto Steps 9B in FIG. 1 e. FIG. 2 shows the NEAR reaction products areeasily distinguished from background products by gel electrophoresis.

Example 2 Detection of RNA NEAR Assay Products by Gel Electrophoresis

The NEAR reaction can also amplify RNA targets. In this case, the targetis Ebola Armored RNA, which is a −600 base strand of RNA encapsulated byMS2 phage coat proteins to simulate a viral particle. The reaction isdesigned to amplify a 25-base region of the Ebola genome containedwithin the encapsulated RNA sequence. Reaction products run on a 20%polyacrylamide gel (FIG. 3) show the amplified 25mer product along with23mer and 20mer background products. This example demonstrates theability of the NEAR reaction to amplify RNA released from virus-likeparticles.

Example 3 Detection of DNA and RNA NEAR Assay Products by MassSpectrometry

The NEAR reaction amplification products can also be detected by massspectrometry using an ESI/TOF system with a front end LC. The reactionproducts observed are multiple charged ion species. Usually, the −3 or−4 charge state is the major peak in the spectrum (in the range of1000-3000 AMU), depending on the length of the oligonucleotide product.The sodium adduct is usually present in the spectrum as a peak adjacentto the major peak at roughly 20-25% the intensity. The unique peaks forthe positive reactions in the presence of target are visible in bothFIGS. 4 and 5 for the DNA and RNA NEAR reactions respectively. Thebackground products formed in these NEAR reactions are not shown in themass range of these spectra.

Example 4 Real-Time Detection of the NEAR Assay Amplification

The NEAR amplification reaction can also be monitored, as shown in FIG.6, in real-time with SYBR II fluorescence. The fluorescence increases asSYBR II intercalates into the amplified double-stranded products. Thebackground products also generate fluorescence at a slower rate than thetrue product. Optimization of amplification sequence, reactiontemperature and reaction buffer conditions are necessary in order tovisualize distinct separation between the positive reactions and thenegative controls.

Example 5 FRET Detection of Real-Time NEAR Assay Amplification

NEAR amplification can also be monitored by Fluorescence ResonanceEnergy Transfer (FRET), as shown in FIG. 7. Amplification occurs usingdual labeled templates, one on each end (5-FAM, 3-BHQ). Fluorescence isgenerated from the FAM-labeled oligonucleotide upon cleavage of thetemplate by the nicking enzyme when it becomes double-stranded. Sincefluorescence is produced by the initial nicking reaction, this detectionmethod is extremely responsive. Since the 3′ ends of the templates areblocked from extension by the quenching label, the production ofbackground fluorescence is inhibited.

Example 6 Molecular Beacon Detection of Real-Time NEAR Amplification

A third method of monitoring real-time amplification is using molecularbeacons, as shown in FIG. 8. In this case, the amplified producthybridizes to the loop region of the molecular beacon resulting in anincrease in fluorescence from the separation of the fluorophore andquencher on each end of the hairpin stem. Since this interaction occurspost-amplification, it is considered pseudo-real-time and can beslightly slower in response relative to the FRET approach.

Example 7 False Alarm Rate Testing

This experiment was designed to probe the probability that the NEARamplification reaction will yield a true product in the negativereaction, or a false positive. NEAR reactions directed at specificamplification of a 25mer region specific to the Bacillus subtilis genomewere run in the presence (n=120) and absence (n=320) of Bacillussubtilis genomic DNA. End point reactions were run on the massspectrometer and the area under the curve (AUC) for the product masspeak in the mass spectrum was analyzed. As shown in FIG. 9, the resultsshow that none of the 320 negative reactions resulted in a falsepositive with AUC values equal to the water control. The true positiveAUC values were at least 3 standard deviations apart from the truenegatives. Overall, these results demonstrate the reproducible nature ofthe NEAR assay.

Example 8 Beacon Detection: NEAR Assay Reproducibility with BeaconDetection

The molecular beacon detection of NEAR reaction products can also beused as an endpoint reading. As shown in FIG. 10, the ratio of NEARreaction products can be manipulated by varying the input ratio of theforward and reverse templates. Skewing the templates to favor one of thereaction products allows the single-stranded product to be available forhybridization to a molecular beacon. The open beacon generates afluorescent signal. This detection method is extremely reproducible. Inthis study, two operators performed replicates of the same assay on twodifferent days. The results of this study demonstrate thereproducibility of the assay from one day to the next as well asreproducibility between operators.

Example 9 NEAR Assay Sensitivity with Beacon Detection

The sensitivity of the NEAR assay with beacon read-out was tested usinga dilution of Francisella tularensis genomic DNA. As shown in FIG. 11,as few as 50 copies were detected above the no target control.

Example 10 Concentration of Amplified Products for NEAR DNAAmplification

The sensitivity of the NEAR assay has also been studied using massspectrometry detection of the reaction products. FIG. 12 shows signalabove the no target control down to 100 copies. The data from this studywas used to correlate the input copy number to the final amount ofamplified product. In this study, the AUC values of the mass specproduct peaks were fit to a standard curve to give the estimated finalconcentration of amplified product for the NEAR assay. The amount ofamplified product ranges from approximately 250 nM to almost 1 μM for1E+2 and 1E+5 copies respectively. This product amount results in a 1E+8to 7E+10-fold amplification. These reactions were performed without thehot-start conditions, in fact hot-start conditions have been shown todramatically increase the amount of product amplified, so a furtherincrease in amplification is achieved. The zero copy amplificationreaction has a positive final concentration due to the y-intercept valuein the standard curve equation.

Example 11 Concentration of Amplified Products for RNA Assay

A similar study was performed on the NEAR amplification of RNA. Adilution of RNA targets were amplified by the NEAR assay. Products wererun on the mass spec and the AUC values of the product peaks wereanalyzed against a standard curve to determine the concentration of thefinal product, as shown in FIG. 13. A 12 minute amplification startingwith 30 and 30,000 copies of initial target results in a 3E+9 to1E+7-fold amplification respectively. The lower extent of amplificationcompared to the DNA amplification could be due to the less efficientreverse transcriptase ability of the polymerase compared to itsreplication abilities. Also, the RNA:DNA hybrid formed upon theextension of the reverse template is a stronger interaction compared toa normal DNA:DNA hybrid and will have less breathing to allow for theforward or another reverse template to displace one strand. However,amplification products from the RNA reaction were detected down to <100copies.

Example 12 NEAR Reaction Specificity for DNA

Since the reaction products are usually between 20 and 30 bases inlength, the question arises as to whether or not these shortamplification assays can be specific enough to target a single sequenceregion with other near neighbor genomes present. The NEAR reaction wastested for its specificity by running the amplification reaction in thepresence and absence of varying amounts of the near neighbor genomic DNA(FIG. 14). In this case, the assay detects a specific sequence in thepX02 plasmid of Bacillus anthracis and the near neighbor genome isBacillus thuringiensis (kurstaki). The reactions were analyzed by theAUC values for the product peaks. The figure below demonstrates that inthe absence of the correct target (Bacillus anthracis), there is no trueproduct amplified (the levels are so low that they are not visible onthe scale of the graph). The amount of amplification of the positivereactions is consistent, with larger error bars for the 0 and 5E+5copies of Bacillus thuringiensis (5E+4 copies of Bacillus anthracis) dueto a single lower value for one of the triplicate runs. Overall theexperiment demonstrates that the NEAR reaction is very specific to thetarget sequence when the assay is designed within a unique region of thegenome.

Example 13 Interferent Testing

A panel of interferents was tested to monitor the effect of each on theNEAR assay amplification. FIG. 15 demonstrates the robust nature of theNEAR assay in the presence of interferents. Some interferents that areknown to inhibit PCR, such as humic acid, did not appear to inhibit theNEAR assay, though the amount of each interferent is unknown. Fromstatistical analysis only interferent B, C, and E were statisticallydifferent from the control assay x. In the B, C, and E cases, thedifference resulted in increased product amplification.

Example 14 Multiplexing of Two Sequences with NEAR DNA Assays

A DNA duplex was designed for capillary electrophoresis (CE) detection.Amplification products were 25 bases (Bacillus anthracis assay, Ba) and27 bases (Bacillus subtilis assay, Bs) in length with backgroundproduction of a 23mer. The reaction was run for 10 minutes in thepresence or absence of 5E+5 copies of the respective genomic DNA target.The samples were run on a 20% polyacrylamide gel to visualize thereaction products. FIG. 16 indicates the presence of positive productamplification when Bacillus subtilis only is present as well as whenboth Bacillus subtilis and Bacillus anthracis are present.

Example 15 NEAR DNA Assay Duplex Specificity

The NEAR DNA duplex reaction with Bacillus subtilis (Bs) and Bacillusanthracis (Ba) was shown to be specific to the respective genomes. Theassays were run in the presence of the near neighbor, Bacillusthuringiensis, as shown in FIG. 17. In the negative reaction where bothtemplate sets are present as well as the Bacillus thuringiensis genomicDNA, there is no product band in the 25 or 27mer region. Product bandsappear only when the specific genomic target is present, whichdemonstrates the specificity of the duplex reaction.

Example 16 Multiplexing with NEAR RNA Assays

An MS2 assay that amplifies a 27mer product and an Ebola assay thatamplifies a 25mer product was developed and multiplexed so that alltemplates are present in each assay and amplification of products isdependent on the target present. This combination of templates formsbackground products that are 23 bases and 20 bases in length. The gelshown in FIG. 18 demonstrates the ability for the NEAR reaction toamplify multiple RNA targets in a single reaction.

Example 17 Amplification from Lysed Spores by NEAR Assay

Amplification was performed on semi-processed samples to determinewhether it is possible to amplify DNA released from spores throughlysis. The negative control reaction contained DNase-treated spores,unlysed, so no DNA should be present to amplify. The positive controlreaction contained purified genomic DNA at concentrations around theamount of DNA estimated to be released through lysis. Results in FIG. 19show that amplification with unlysed DNase-treated spores results in noproduct amplification as expected, whereas the three samples lysedbefore amplification resulted in product amounts in the range of thetheoretical amounts.

Example 18 Capture and Extension

The NEAR reaction products can also be detected on a solid surface. Acapture probe attached at the 5′ end to the surface through abiotin/streptavidin attachment can bind to the reaction products fromwhich polymerase extends to form a stable duplex that SYBR and anyintercalating dye can detect. The capture probe is designed to favorextension through binding to the true product over background productsbecause the 3′ base of the capture probe is complementary to the middlespacer base in the product which is not present in either of thetemplates or the background products. FIG. 20 demonstrates the increasedfluorescence of the NEAR products in the presence of the capture probeand polymerase over the average binding (same reaction in the absence ofpolymerase, to preclude extension of the capture probe) and the notarget control where only background products are amplified, but cannotform a stable duplex with the capture probe for polymerase to extend.

Example 19 Surface NEAR FRET DNA Assay

The NEAR reaction can also be performed with the templates immobilizedon the surface. The templates for FRET detection of surfaceamplification usually have three modifications: one 5′ biotin with a TEGspacer, one FAM fluorophore internal to the biotin, and a quencher onthe 3′ end which serves to block background amplification as well as toquench the FAM fluorophore. The template is immobilized on the surfacethrough biotin/streptavidin attachment. FIG. 21 demonstrates that withboth templates immobilized along with additional mixing, the reactionproceeds at a much slower rate than the solution amplification rate(amplification in 16 minutes for 1E+6 copies of genomic DNA). When asingle template is immobilized on the surface and the other template isfree in solution, the amplification reaction is increased to 10 minutedetection for 1E+6 copies of genomic DNA. Fluorescence from backgroundproducts is observed −3.5 minutes after the product signal, similar towhat is observed for solution phase kinetics, but slowed considerably.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Singular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “asubset” includes a plurality of such subsets, reference to “a nucleicacid” includes one or more nucleic acids and equivalents thereof knownto those skilled in the art, and so forth. The term “or” is not meant tobe exclusive to one or the terms it designates. For example, as it isused in a phrase of the structure “A or B” may denote A alone, B alone,or both A and B.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andsystems similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the methods, devices,and materials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of describing anddisclosing the processes, systems, and methodologies that are reportedin the publications which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Modifications may be made to the foregoing without departing from thebasic aspects of the invention. Although the invention has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, and yet these modifications and improvements are within thescope and spirit of the invention. The invention illustrativelydescribed herein suitably may be practiced in the absence of anyelement(s) not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. Thus, the terms and expressions which have been employed are usedas terms of description and not of limitation, equivalents of thefeatures shown and described, or portions thereof, are not excluded, andit is recognized that various modifications are possible within thescope of the invention. Embodiments of the invention are set forth inthe following claims.

1.-66. (canceled)
 67. A method of amplifying, comprising: preparing amixture comprising: (i) a target nucleic acid present in a sampleobtained from an animal, the target nucleic acid having a targetpolynucleotide sequence, (ii) a polymerase, (iii) a nicking enzyme, (iv)a first oligonucleotide comprising a nicking site and a nicking enzymebinding site, and (v) a second oligonucleotide comprising a nicking siteand a nicking enzyme binding site.
 68. The method of claim 67, whereinthe target polynucleotide sequence is amplified from steps comprising:(a) forming a first duplex comprising the target polynucleotide sequenceand the first oligonucleotide; (b) extending, using the polymerase, thefirst oligonucleotide along the target polynucleotide sequence to forman extended first oligonucleotide comprising a sequence complementary tothe second oligonucleotide; (c) forming a second duplex comprising thesecond oligonucleotide and the extended first oligonucleotide; (d)extending, using the polymerase, the second oligonucleotide along theextended first oligonucleotide to form an extended secondoligonucleotide comprising a sequence complementary to the firstoligonucleotide and a first double-stranded nicking enzyme binding site;(e) nicking, with the nicking enzyme, at the nicking site on the firstoligonucleotide to produce a first polynucleotide fragment; (f)extending, using the polymerase, the first polynucleotide fragment alongthe extended second oligonucleotide to produce a double-stranded nucleicacid product and a second double-stranded nicking enzyme binding site.69. The method of claim 68, wherein the double-stranded nucleic acidproduct comprises: i) a first strand and a second strand, wherein thefirst strand comprises a first polynucleotide sequence corresponding tothe target polynucleotide sequence and the second strand comprises asecond polynucleotide sequence complementary to the targetpolynucleotide sequence, and ii) first and second double-strandednicking sites spaced apart by the target polynucleotide sequence. 70.The method of claim 68, further comprising the steps of: a) nicking,using the nicking enzyme, the first nicking site of the double-strandednucleic acid product to prepare a first polynucleotide fragment andnicking, using the nicking enzyme, the second nicking site of thedouble-stranded nucleic acid product to produce a second polynucleotidefragment; b) extending, using the polymerase, a portion of the firstpolynucleotide fragment to produce a first product polynucleotide andextending, using a polymerase, a portion of the second polynucleotidefragment to produce a second product polynucleotide; and c) nicking,using the nicking enzyme, the first product polynucleotide to release acopy of the first polynucleotide sequence and nicking, using the nickingenzyme, the second product polynucleotide to release a copy of thesecond polynucleotide sequence.
 71. The method of claim 67, wherein theanimal is a human.
 72. The method of claim 67, wherein the targetnucleic acid is obtained from an animal pathogen.
 73. The method ofclaim 72, wherein the animal pathogen is a single-stranded DNA virus,double-stranded DNA virus, or single-stranded RNA virus.
 74. The methodof claim 72, wherein the animal pathogen is a bacterium.
 75. The methodof claim 72, wherein the animal pathogen contains spores and the targetpolynucleotide is amplified from the spores without the need for lysisof the spores.
 76. The method of claim 67, wherein the sample obtainedfrom an animal is obtained from the blood, bone marrow, mucus, lymph,hard tissues (e.g. liver, spleen, kidney, lung or ovary), biopsies,sputum, saliva, tears, faeces or urine of the animal.
 77. The method ofclaim 76, wherein the sample obtained from an animal is obtained fromthe mucus, sputum, or saliva of the animal.
 78. The method of claim 67,wherein the target nucleic acid is double-stranded DNA.
 79. The methodof claim 67, wherein the target nucleic acid is single-stranded DNA. 80.The method of claim 67, wherein the target nucleic acid is RNA.
 81. Themethod of claim 67, wherein the target nucleic acid is selected from thegroup consisting of genomic DNA, plasmid DNA, viral DNA, mitochondrialDNA, cDNA, synthetic double-stranded DNA and synthetic single-strandedDNA.
 82. The method of claim 81, wherein the target nucleic acid isgenomic DNA.
 83. The method of claim 81, wherein the target nucleic acidis viral DNA.
 84. The method of claim 67, which is performed without aninitial heat denaturation step.
 85. The method of claim 67, wherein thenicking enzyme is Nt.BstNBI.
 86. The method of claim 67, wherein thenicking enzyme does not nick within the target polynucleotide sequence.87. The method of claim 67, which is performed without the use oftemperature cycling.
 88. The method of claim 67, which is performed atabout 55° C.-59° C.
 89. The method of claim 67, which is performed at aconstant temperature for about 1 to 20 minutes.
 90. The method of claim68, which is performed at a temperature higher than the meltingtemperature of the first oligonucleotide/target polynucleotide sequencecomplex.
 91. The method of claim 67, further comprising detectingamplification product.
 92. The method of claim 91, wherein theamplification product is detected by a detection method selected fromthe group consisting of gel electrophoresis, mass spectrometry, SYBR Ifluorescence, SYBR II fluorescence, SYBR Gold, Pico Green, TOTO-3,intercalating dye detection, fluorescence resonance energy transfer(FRET), molecular beacon detection, surface capture, capillaryelectrophoresis, incorporation of labeled nucleotides to allow detectionby capture, fluorescence polarization, and lateral flow capture, or acombination thereof.
 93. The method of claim 67, wherein the targetpolynucleotide sequence is amplified 1E+9-fold or more in about fiveminutes.
 94. A method of amplifying, comprising: preparing a mixturecomprising: (i) a target nucleic acid present in a sample obtained froman animal, the target nucleic acid having a target polynucleotidesequence, (ii) a polymerase, (iii) a nicking enzyme, (iv) a firstoligonucleotide comprising a nicking site and a nicking enzyme bindingsite, and (v) a second oligonucleotide comprising a nicking site and anicking enzyme binding site; which method is performed without aninitial heat denaturation step.
 95. A method of amplifying, comprising:preparing a mixture comprising: (i) genomic DNA present in a sampleobtained from an animal, the genomic DNA having a target polynucleotidesequence, (ii) a polymerase, (iii) a nicking enzyme, (iv) a firstoligonucleotide comprising a nicking site and a nicking enzyme bindingsite, and (v) a second oligonucleotide comprising a nicking site and anicking enzyme binding site; which method is performed without aninitial heat denaturation step.